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1.  Risk Factors for Pandemic (H1N1) 2009 Seroconversion among Adults, Singapore, 2009 
Emerging Infectious Diseases  2011;17(8):1455-1462.
PMCID: PMC3381584  PMID: 21801623
Influenza A virus; pandemics; seroepidemiologic study; cohort analysis; pandemic (H1N1) 2009; influenza; risk factors; travel; public transport; viruses; Singapore; adults; research
2.  Pandemic (H1N1) 2009 Surveillance and Prevalence of Seasonal Influenza, Singapore 
Emerging Infectious Diseases  2010;16(1):103-105.
On April 25, 2009, Singapore implemented strict containment measures for pandemic (H1N1) 2009 with enhanced surveillance and hospital isolation. In the first month, seasonal influenza, predominantly virus subtype H3N2, was diagnosed for 32% of patients with acute febrile respiratory illness. Our findings underscore the high prevalence of seasonal influenza in Singapore.
PMCID: PMC2874379  PMID: 20031051
Pandemic (H1N1) 2009; seasonal influenza; influenza; surveillance; viruses; Singapore; expedited; dispatch
3.  Early clinical and laboratory risk factors of intensive care unit requirement during 2004–2008 dengue epidemics in Singapore: a matched case–control study 
BMC Infectious Diseases  2014;14(1):649.
Dengue infection can result in severe clinical manifestations requiring intensive care. Effective triage is critical for early clinical management to reduce morbidity and mortality. However, there is limited knowledge on early risk factors of intensive care unit (ICU) requirement. This study aims to identify early clinical and laboratory risk factors of ICU requirement at first presentation in hospital and 24 hours prior to ICU requirement.
A retrospective 1:4 matched case–control study was performed with 27 dengue patients who required ICU, and 108 dengue patients who did not require ICU from year 2004–2008, matched by year of dengue presentation. Univariate and multivariate conditional logistic regression were performed. Optimal predictive models were generated with statistically significant risk factors identified using stepwise forward and backward elimination method.
ICU dengue patients were significantly older (P=0.003) and had diabetes (P=0.031), compared with non-ICU dengue patients. There were seven deaths among ICU patients at median seven days post fever. At first presentation, the WHO 2009 classification of dengue severity was significantly associated (P<0.001) with ICU, but not the WHO 1997 classification. Early clinical risk factors at presentation associated with ICU requirement were hematocrit change ≥20% concurrent with platelet <50 K [95% confidence-interval (CI)=2.46-30.53], hypoproteinemia (95% CI=1.09-19.74), hypotension (95% CI=1.83-31.79) and severe organ involvement (95% CI=3.30-331). Early laboratory risk factors at presentation were neutrophil proportion (95% CI=1.04-1.17), serum urea (95% CI=1.02-1.56) and alanine aminotransferase level (95% CI=1.001-1.06). This predictive model has sensitivity and specificity up to 88%. Early laboratory risk factors at 24 hours prior to ICU were lymphocyte (95% CI=1.03-1.38) and monocyte proportions (95% CI=1.02-1.78), pulse rate (95% CI=1.002-1.14) and blood pressure (95% CI=0.92-0.996). This predictive model has sensitivity and specificity up to 88.9% and 78%, respectively.
This is the first matched case–control study, to our best knowledge, that identified early clinical and laboratory risk factors of ICU requirement during hospitalization. These factors suggested differential pathophysiological background of dengue patients as early as first presentation prior to ICU requirement, which may reflect the pathogenesis of dengue severity. These risk models may facilitate clinicians in triage of patients, after validating in larger independent studies.
Electronic supplementary material
The online version of this article (doi:10.1186/s12879-014-0649-2) contains supplementary material, which is available to authorized users.
PMCID: PMC4267742  PMID: 25475217
Dengue; Intensive care unit; Early risk factors; Matched case–control
4.  Rate of decline of antibody titers to pandemic influenza A (H1N1-2009) by hemagglutination inhibition and virus microneutralization assays in a cohort of seroconverting adults in Singapore 
BMC Infectious Diseases  2014;14(1):414.
The rate of decline of antibody titers to influenza following infection can affect results of serological surveys, and may explain re-infection and recurrent epidemics by the same strain.
We followed up a cohort who seroconverted on hemagglutination inhibition (HI) antibody titers (≥4-fold increase) to pandemic influenza A(H1N1)pdm09 during a seroincidence study in 2009. Along with the pre-epidemic sample, and the sample from 2009 with the highest HI titer between August and October 2009 (A), two additional blood samples obtained in April 2010 and September 2010 (B and C) were assayed for antibodies to A(H1N1)pdm09 by both HI and virus microneutralization (MN) assays. We analyzed pair-wise mean-fold change in titers and the proportion with HI titers ≥ 40 and MN ≥ 160 (which correlated with a HI titer of 40 in our assays) at the 3 time-points following seroconversion.
A total of 67 participants contributed 3 samples each. From the highest HI titer in 2009 to the last sample in 2010, 2 participants showed increase in titers (by HI and MN), while 63 (94%) and 49 (73%) had reduction in HI and MN titers, respectively. Titers by both assays decreased significantly; while 70.8% and 72.3% of subjects had titers of ≥ 40 and ≥ 160 by HI and MN in 2009, these percentages decreased to 13.9% and 36.9% by September 2010. In 6 participants aged 55 years and older, the decrease was significantly greater than in those aged below 55, so that none of the elderly had HI titers ≥ 40 nor MN titers ≥ 160 by the final sample. Due to this decline in titers, only 23 (35%) of the 65 participants who seroconverted on HI in sample A were found to seroconvert between the pre-epidemic sample and sample C, compared to 53 (90%) of the 59 who seroconverted on MN on Sample A.
We observed marked reduction in titers 1 year after seroconversion by HI, and to a lesser extent by MN. Our findings have implications for re-infections, recurrent epidemics, vaccination strategies, and for cohort studies measuring infection rates by seroconversion.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2334-14-414) contains supplementary material, which is available to authorized users.
PMCID: PMC4133624  PMID: 25066592
Pandemic influenza A(H1N1)pdm09; Declining antibody; Seroconversion; Hemagglutination inhibition assay; Microneutralization assay
5.  Predictive Tools for Severe Dengue Conforming to World Health Organization 2009 Criteria 
Dengue causes 50 million infections per year, posing a large disease and economic burden in tropical and subtropical regions. Only a proportion of dengue cases require hospitalization, and predictive tools to triage dengue patients at greater risk of complications may optimize usage of limited healthcare resources. For severe dengue (SD), proposed by the World Health Organization (WHO) 2009 dengue guidelines, predictive tools are lacking.
We undertook a retrospective study of adult dengue patients in Tan Tock Seng Hospital, Singapore, from 2006 to 2008. Demographic, clinical and laboratory variables at presentation from dengue polymerase chain reaction-positive and serology-positive patients were used to predict the development of SD after hospitalization using generalized linear models (GLMs).
Principal findings
Predictive tools compatible with well-resourced and resource-limited settings – not requiring laboratory measurements – performed acceptably with optimism-corrected specificities of 29% and 27% respectively for 90% sensitivity. Higher risk of severe dengue (SD) was associated with female gender, lower than normal hematocrit level, abdominal distension, vomiting and fever on admission. Lower risk of SD was associated with more years of age (in a cohort with an interquartile range of 27–47 years of age), leucopenia and fever duration on admission. Among the warning signs proposed by WHO 2009, we found support for abdominal pain or tenderness and vomiting as predictors of combined forms of SD.
The application of these predictive tools in the clinical setting may reduce unnecessary admissions by 19% allowing the allocation of scarce public health resources to patients according to the severity of outcomes.
Author Summary
Dengue is a mosquito-borne tropical disease that poses a large economic and health burden to 40% of the world's population. Some dengue patients can evolve into a more severe form of the illness (severe dengue) and ideally should be hospitalized when this occurs. However, while only a small proportion of patients have severe dengue, many other patients are unnecessarily hospitalized. Being able to identify those patients at the highest risk of developing severe dengue can be very useful to reduce costs to hospitals and patients. In this study, we develop predictive tools to identify those patients that should be hospitalized. We find that some common features such as age, gender, fever duration, fever on admission, vomiting or abdominal distension can be good predictors of severe dengue. This is important because these features are easy to identify even in resource-limited settings where laboratory tests are not widely available.
PMCID: PMC4091876  PMID: 25010515
6.  Spatial epidemiology and climatic predictors of paediatric dengue infections captured via sentinel site surveillance, Phnom Penh Cambodia 2011–2012 
BMC Public Health  2014;14:658.
Dengue is a major contributor to morbidity in children aged twelve and below throughout Cambodia; the 2012 epidemic season was the most severe in the country since 2007, with more than 42,000 reported (suspect or confirmed) cases.
We report basic epidemiological characteristics in a series of 701 patients at the National Paediatric Hospital in Cambodia, recruited during a prospective clinical study (2011–2012). To more fully explore this cohort, we examined climatic factors using multivariate negative binomial models and spatial clustering of cases using spatial scan statistics to place the clinical study within a larger epidemiological framework.
We identify statistically significant spatial clusters at the urban village scale, and find that the key climatic predictors of increasing cases are weekly minimum temperature, median relative humidity, but find a negative association with rainfall maximum, all at lag times of 1–6 weeks, with significant effects extending to 10 weeks.
Our results identify clustering of infections at the neighbourhood scale, suggesting points for targeted interventions, and we find that the complex interactions of vectors and climatic conditions in this setting may be best captured by rising minimum temperature, and median (as opposed to mean) relative humidity, with complex and limited effects from rainfall. These results suggest that real-time cluster detection during epidemics should be considered in Cambodia, and that improvements in weather data reporting could benefit national control programs by allow greater prioritization of limited health resources to both vulnerable populations and time periods of greatest risk. Finally, these results add to the increasing body of knowledge suggesting complex interactions between climate and dengue cases that require further targeted research.
PMCID: PMC4085229  PMID: 24972712
7.  A prospective observational study of the prevalence and risk factors for colonization by antibiotic resistant bacteria in patients at admission to hospital in Singapore 
BMC Infectious Diseases  2014;14:298.
Drug resistant organisms pose an increasing threat to the successful treatment of common infections. Understanding colonization patterns of these bacteria is important for effective antibiotic treatment and infection control guidelines.
A prospective observational study was performed to determine the prevalence of colonization with extended-spectrum β-lactamase-producing Enterobacteriaceae (ESBL-E), methicillin-resistant Staphylococcus aureus (MRSA), and vancomycin-resistant Enterococcus (VRE) among patients admitted via the emergency department to a public tertiary hospital in Singapore. Anterior nares, groin, axillary and rectal swabs were collected at admission and cultured using standard bacteriological techniques. Clinical data including healthcare contact within the past 12 months and recent antibiotic use was collected and analyzed using a logistic regression model.
1006 patients were screened. 124 (12.4%) were colonized by ESBL-E, 18 (1.8%) by MRSA while no VRE was detected. Antibiotic use within the past month was the only significant predictor for ESBL-E colonization in the regression model, with an adjusted odds ratio (AOR) of 2.58 (1.04 to 6.42). In participants recently prescribed antibiotics and hospitalized in the previous 3 months, 29.4% were colonized by ESBL-E. This represented 20.2% of the total ESBL-E burden, and ESBL-E was also detected in 6.3% of participants with no healthcare contact. Hospitalization and outpatient hospital visits predicted MRSA colonization in the univariate analysis. Neither was statistically significant in the logistic regression model, with AORs for MRSA colonization following hospitalization in the past 3 and 12 months of 3.81 [95% CI 0.84-17.28] and 3.48 [0.64-18.92] respectively.
A high prevalence of colonization with ESBL-E was evident among patients at admission, even in the absence of recent antibiotic use or contact with healthcare.
PMCID: PMC4057577  PMID: 24889720
Antibiotics; Colonization; ESBL; Fecal carriage; MRSA
8.  Predictive Value of Proteinuria in Adult Dengue Severity 
Dengue is an important viral infection with different presentations. Predicting disease severity is important in triaging patients requiring hospital care. We aim to study the value of proteinuria in predicting the development of dengue hemorrhagic fever (DHF), utility of urine dipstick test as a rapid prognostic tool.
Methodology and principal findings
Adult patients with undifferentiated fever (n = 293) were prospectively enrolled at the Infectious Disease Research Clinic at Tan Tock Seng Hospital, Singapore from January to August 2012. Dengue infection was confirmed in 168 (57%) by dengue RT-PCR or NS1 antigen detection. Dengue cases had median fever duration of 6 days at enrolment. DHF was diagnosed in 34 cases according to the WHO 1997 guideline. Dengue fever (DF) patients were predominantly younger and were mostly seen in the outpatient setting with higher platelet level. Compared to DF, DHF cases had significantly higher peak urine protein creatinine ratio (UPCR) during clinical course (26 vs. 40 mg/mmol; p<0.001). We obtained a UPCR cut-off value of 29 mg/mmol based on maximum AUC in ROC curves of peak UPCR for DF versus DHF, corresponding to 76% sensitivity and 60% specificity. Multivariate analysis with other readily available clinical and laboratory variables increased the AUC to 0.91 with 92% sensitivity and 80% specificity. Neither urine dipstick at initial presentation nor peak urine dipstick value during the entire illness was able to discriminate between DF and DHF.
Proteinuria measured by a laboratory-based UPCR test may be sensitive and specific in prognosticating adult dengue patients.
Author Summary
Dengue infection is getting more common in recent years, affecting all age groups. Currently, there is no specific treatment for dengue. Close clinical monitoring and careful fluid therapy is the only way of management for those with severe dengue disease, i.e., dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS). It is crucial to know among patients with dengue, who will potentially progress to DHF/DSS in the most reliable, economical and fastest way, so as to prioritise limited resources. We investigated the presence of protein in urine as an indicator of progression to DHF/DSS. Adult patients with dengue were enrolled to our clinic. Clinical data, blood and urine were collected. We found that patients who were going to develop DHF/DSS had increased protein one day prior to developing to DHF/DSS. DHF/DSS cases had higher protein levels in urine compared to patients with just dengue fever. Laboratory-based urine protein data, when used together with other readily available blood tests, helped to detect 92% of DHF cases correctly. Currently available clinic-based urine protein test strip was not useful in predicting severe disease. Future studies may improve the ability of the clinic-based tests, thus reducing the reliance on laboratory testing.
PMCID: PMC3930505  PMID: 24587464
9.  Utility of warning signs in guiding admission and predicting severe disease in adult dengue 
BMC Infectious Diseases  2013;13:498.
The recommendation from the 2009 World Health Organization guidelines for managing dengue suggests that patients with any warning sign can be hospitalized for observation and management. We evaluated the utility of using warning signs to guide hospital admission and predict disease progression in adults.
We conducted a prospective cohort study from January 2010 to September 2012. Daily demographic, clinical and laboratory data were collected from adult dengue patients. Warning signs were recorded. The proportion of admitted patients using current admission criteria and warning signs was compared. The sensitivity, specificity, positive and negative predictive values of warning signs in predicting disease progression were also evaluated.
Four hundred and ninety-nine patients with confirmed dengue were analyzed. Using warning signs instead of the current admission criteria will lead to a 44% and 31% increase in admission for DHF II-IV and SD cases respectively. The proportion of non-severe dengue cases which were admitted also increased by 32% for non DHF II-IV and 33% for non-SD cases. Absence of any warning signs had a NPV of 91%, 100% and 100% for DHF I-IV, DHF II-IV and SD. Of those who progressed to severe illness, 16.3% had warning signs on the same day while 51.3% had warning signs the day before developing severe illness, respectively.
Our findings demonstrated that patients without any warning signs can be managed safely with ambulatory care to reduce hospital resource burden. No single warning sign can independently predict disease progression. The window from onset of warning sign to severe illness in most cases was within one day.
PMCID: PMC4015176  PMID: 24152678
Dengue; Warning signs; Utility; Disease progression; Admission
10.  High prevalence of CXCR4 usage among treatment-naive CRF01_AE and CRF51_01B-infected HIV-1 subjects in Singapore 
Recent studies suggest HIV-1 inter-subtype differences in co-receptor usage. We examined the correlation between HIV-1 subtype and co-receptor usage among treatment-naïve HIV-1 subjects in Singapore. Additionally, we investigated whether the subtype co-receptor association was influenced by stage of infection.
V3 sequences of HIV-1 envelope protein gp120 were obtained from 110 HIV treatment-naïve patients and genotypic co-receptor tropism determination was performed using Geno2pheno. Two false-positive rate (FPR) cut-offs, 10% and 5.75% were selected for tropism testing.
Subtype assignment of viral strains from 110 HIV-infected individuals based on partial sequencing of HIV-1 pol, gp120 and gp41 were as follows: 27 subtype B, 64 CRF01_AE, 10 CRF51_01B, and 9 other subtypes. At FPR=10%, 10 (100%) CRF51_01B-infected subjects and 26 (40.6%) CRF01_AE-infected subjects had CXCR4-using virus, compared to 7 (25.9%) subtype B subjects and 1 (11.1%) CRF33_01B-infected subject (P < 0.001). At FPR=5.75%, 10 (100%) CRF51_01B-infected subjects and 20 (31.3%) CRF01_AE-infected subjects had CXCR4-using virus, compared to 4 (14.8%) subtype B and 1 (11.1%) CRF33_01B-infected subjects (P < 0.001). Among those with evidence of seroconversion within 2 years prior to study enrolment, 100% of CRF51_01B-infected subjects had CXCR4-using virus, independent of Geno2pheno FPR.
CRF51_01B and CRF01_AE-infected individuals have higher prevalence of CXCR4-usage compared to subtype B infected individuals. Further studies examining these differences could help optimise the use of CCR5-antagonist in populations with these subtypes, and increase our understanding of HIV-1 biology.
PMCID: PMC3585921  PMID: 23421710
CXCR4 usage; HIV-1; treatment-naïve
11.  Utilities and Limitations of the World Health Organization 2009 Warning Signs for Adult Dengue Severity 
In 2009, the World Health Organization (WHO) proposed seven warning signs (WS) as criteria for hospitalization and predictors of severe dengue (SD). We assessed their performance for predicting dengue hemorrhagic fever (DHF) and SD in adult dengue.
DHF, WS and SD were defined according to the WHO 1997 and 2009 dengue guidelines. We analyzed the prevalence, sensitivity (Sn), specificity (Sp), positive predictive value (PPV) and negative predictive value (NPV) of WS before DHF and SD onset.
Of 1507 cases, median age was 35 years (5th–95th percentile, 17–60), illness duration on admission 4 days (5th–95th percentile, 2–6) and length of hospitalization 5 days (5th–95th percentile, 3–7). DHF occurred in 298 (19.5%) and SD in 248 (16.5%). Of these, WS occurred before DHF in 124 and SD in 65 at median of two days before DHF or SD. Three commonest warning signs were lethargy, abdominal pain/tenderness and mucosal bleeding. No single WS alone or combined had Sn >64% in predicting severe disease. Specificity was >90% for both DHF and SD with persistent vomiting, hepatomegaly, hematocrit rise and rapid platelet drop, clinical fluid accumulation, and any 3 or 4 WS. Any one of seven WS had 96% Sn but only 18% Sp for SD.
No WS was highly sensitive in predicting subsequent DHF or SD in our confirmed adult dengue cohort. Persistent vomiting, hepatomegaly, hematocrit rise and rapid platelet drop, and clinical fluid accumulation, as well as any 3 or 4 WS were highly specific for DHF or SD.
Author Summary
Dengue is a mosquito-borne infection with significant public health burden in tropical and subtropical regions. Clinical presentations may vary from self-limiting fever to severe dengue including death. The World Health Organization 2009 dengue guidelines classified dengue into dengue with and without warning signs, and severe dengue. In our adult dengue cohort, we found that lethargy, abdominal pain/tenderness, and mucosal bleeding occurred commonly. As predictors of severe dengue, these were not as specific as persistent vomiting, hepatomegaly, hematocrit rise and rapid platelet drop, and clinical fluid accumulation which occurred infrequently. Presence of any one warning sign had high sensitivity, but low specificity for severe dengue. Removal of lethargy from the list increased the specificity of any one warning sign, but sensitivity decreased. Consideration of these findings may avoid over hospitalization of potentially non-severe dengue patients and reduce burden on the healthcare system.
PMCID: PMC3547865  PMID: 23350013
12.  Prevalence of and risk factors for MRSA colonization in HIV-positive outpatients in Singapore 
Whilst there have been studies on the risks and outcomes of MRSA colonization and infections in HIV-positive patients, local data is limited on the risk factors for MRSA colonization among these patients. We undertook this study in a tertiary HIV care centre to document the risk factors for colonization and to determine the prevalence of MRSA colonization among HIV-positive outpatients in Singapore.
This was a cross-sectional study in which factors associated with MRSA positivity among patients with HIV infection were evaluated. A set of standardized questionnaire and data collection forms were available to interview all recruited patients. Following the interview, trained nurses collected swabs from the anterior nares/axilla/groin (NAG), throat and peri-anal regions. Information on demographics, clinical history, laboratory results and hospitalization history were retrieved from medical records.
MRSA was detected in swab cultures from at least 1 site in 15 patients (5.1%). Inclusion of throat and/or peri-anal swabs increased the sensitivity of NAG screening by 20%. Predictors for MRSA colonization among HIV-positive patients were age, history of pneumonia, lymphoma, presence of a percutaneous device within the past 12 months, history of household members hospitalized more than two times within the past 12 months, and a most recent CD4 count less than 200.
This study highlights that a proportion of MRSA carriers would have been undetected without multiple-site screening cultures. This study could shed insight into identifying patients at risk of MRSA colonization upon hospital visit and this may suggest that a risk factor-based approach for MRSA surveillance focusing on high risk populations could be considered.
PMCID: PMC3540004  PMID: 23126233
Methicillin-resistant Staphylococcus auerus; HIV; Risk factors; Colonization
13.  Simple Clinical and Laboratory Predictors of Chikungunya versus Dengue Infections in Adults 
Dengue and chikungunya are co-circulating vector-borne diseases with substantial overlap in clinical presentations. It is important to differentiate between them during first presentation as their management, especially for dengue hemorrhagic fever (DHF), is different. This study compares their clinical presentation in Singapore adults to derive predictors to assist doctors in diagnostic decision-making.
We compared 117 patients with chikungunya infection diagnosed with reverse transcription-polymerase chain reaction (RT-PCR) with 917 dengue RT-PCR-positive adult patients (including 55 with DHF). We compared dengue fever (DF), DHF, and chikungunya infections by evaluating clinical characteristics of dengue and chikungunya; developing classification tools via multivariate logistic regression models and classification trees of disease etiology using clinical and laboratory factors; and assessing the time course of several clinical variables.
At first presentation to hospital, significantly more chikungunya patients had myalgia or arthralgia, and fewer had a sore throat, cough (for DF), nausea, vomiting, diarrhea, abdominal pain, anorexia or tachycardia than DF or DHF patients. From the decision trees, platelets <118×109/L was the only distinguishing feature for DF versus chikungunya with an overall correct classification of 89%. For DHF versus chikungunya using platelets <100×109/L and the presence of bleeding, the overall correct classification was 98%. The time course analysis supported platelet count as the key distinguishing variable.
There is substantial overlap in clinical presentation between dengue and chikungunya infections, but simple clinical and laboratory variables can predict these infections at presentation for appropriate management.
Author Summary
Dengue and chikungunya are mosquito-borne diseases that are found in similar geographical areas and present with similar symptoms. As their treatment is different, especially for dengue haemorraghic fever (DHF) which is a more severe form of dengue, it is important for healthcare workers to differentiate between them. We studied 117 chikungunya and 917 dengue adult patients (including 55 with DHF) by comparing their clinical presentation and developed decision trees to classify them using simple symptoms and laboratory tests. From the study, we found that at their first appearance in hospital, more chikungunya patients had muscle or joint pains, and fewer had a sore throat, cough, nausea, vomiting, diarrhea, stomach pain, loss of appetite or fast heart beat than DF or DHF patients. From the decision trees, of DF versus chikungunya using only platelet level cut-offs, we could correctly classify 89% of the cases. For DHF versus chikungunya using platelet level cut-offs and the presence of bleeding, the correct classification was 98%. The use of these simple decision trees can therefore predict the subsequent development of these infections for appropriate treatment.
PMCID: PMC3459852  PMID: 23029573
14.  Confirmed adult dengue deaths in Singapore: 5-year multi-center retrospective study 
BMC Infectious Diseases  2011;11:123.
Dengue re-emerges in Singapore despite decades of effective vector control; the infection predominantly afflicts adults. Severe dengue not fulfilling dengue hemorrhagic fever (DHF) criteria according to World Health Organization (WHO) 1997 guideline was increasingly reported. A new WHO 2009 guideline emphasized warning signs and a wider range of severe dengue manifestations. We aim to evaluate the utility of these two guidelines in confirmed adult dengue fatalities.
We conducted a multi-center retrospective chart review of all confirmed adult dengue deaths in Singapore from 1 January 2004 to 31 December 2008.
Of 28 adult dengue deaths, median age was 59 years. Male gender comprised 67.9% and co-morbidities existed in 75%. From illness onset, patients presented for admission at a median of 4 days and death occurred at a median of 12 days. Intensive care admission was required in 71.4%. Probable dengue was diagnosed in 32.1% by WHO 1997 criteria and 78.6% by WHO 2009. The earliest warning sign was persistent vomiting at a median of 1.5 days. Hematocrit change ≥20% concurrent with platelet count <20 × 10^9/L was associated with the shortest interval to death at a median of 3 days. Only 35.7% of death cases fulfilled DHF criteria by WHO 1997 versus severe dengue in 100.0% by WHO 2009 criteria. Deaths were due to shock and organ failure. Acute renal impairment occurred in 71.4%, impaired consciousness 57.1% and severe hepatitis 53.6%.
In our adult fatal dengue cohort, WHO 2009 criteria had higher sensitivity in diagnosing probable dengue and severe dengue compared with WHO 1997. As warning signs, persistent vomiting occurred early and hematocrit change ≥20% concurrent with platelet count <20 × 10^9/L preceded death most closely.
PMCID: PMC3112097  PMID: 21569427
15.  IL-1β, IL-6, and RANTES as Biomarkers of Chikungunya Severity 
PLoS ONE  2009;4(1):e4261.
Little is known about the immunopathogenesis of Chikungunya virus. Circulating levels of immune mediators and growth factors were analyzed from patients infected during the first Singaporean Chikungunya fever outbreak in early 2008 to establish biomarkers associated with infection and/or disease severity.
Methods and Findings
Adult patients with laboratory-confirmed Chikungunya fever infection, who were referred to the Communicable Disease Centre/Tan Tock Seng Hospital during the period from January to February 2008, were included in this retrospective study. Plasma fractions were analyzed using a multiplex-microbead immunoassay. Among the patients, the most common clinical features were fever (100%), arthralgia (90%), rash (50%) and conjunctivitis (40%). Profiles of 30 cytokines, chemokines, and growth factors were able to discriminate the clinical forms of Chikungunya from healthy controls, with patients classified as non-severe and severe disease. Levels of 8 plasma cytokines and 4 growth factors were significantly elevated. Statistical analysis showed that an increase in IL-1β, IL-6 and a decrease in RANTES were associated with disease severity.
This is the first comprehensive report on the production of cytokines, chemokines, and growth factors during acute Chikungunya virus infection. Using these biomarkers, we were able to distinguish between mild disease and more severe forms of Chikungunya fever, thus enabling the identification of patients with poor prognosis and monitoring of the disease.
PMCID: PMC2625438  PMID: 19156204
16.  Clinical and epidemiological predictors of transmission in Severe Acute Respiratory Syndrome (SARS) 
Only a minority of probable SARS cases caused transmission. We assess if any epidemiological or clinical factors in SARS index patients were associated with increased probability of transmission.
We used epidemiological and clinical data on probable SARS patients admitted to Tan Tock Seng Hospital. Using a case-control approach, index patients who had probable SARS who subsequently transmitted the disease to at least one other patient were analysed as "cases" against patients with no transmission as "controls", using multivariate logistic regression analysis.
98 index patients were available for analysis (22 with transmission, 76 with no transmission). Covariates positively associated with transmission in univariate analysis at p < 0.05 included delay to isolation (Day 7 of illness or later), admission to a non-isolation facility, pre-existing chronic respiratory disease and immunosuppressive disease, need for oxygen, shortness of breath, vomiting, and higher lactate dehydrogenase levels and higher neutrophil counts. In the multivariate analysis, only three factors were significant: delay to isolation, admission to a non-isolation facility and higher lactate dehydrogenase levels of >650 IU/L (OR 6.4, 23.8 and 4.7 respectively).
Clinical and epidemiological factors can help us to explain why transmission was observed in some instances but not in others.
PMCID: PMC1624840  PMID: 17049088
17.  Sero-Prevalence and Cross-Reactivity of Chikungunya Virus Specific Anti-E2EP3 Antibodies in Arbovirus-Infected Patients 
Chikungunya virus (CHIKV) and clinically-related arboviruses cause large epidemics with serious economic and social impact. As clinical symptoms of CHIKV infections are similar to several flavivirus infections, good detection methods to identify CHIKV infection are desired for improved treatment and clinical management. The strength of anti-E2EP3 antibody responses was explored in a longitudinal study on 38 CHIKV-infected patients. We compared their anti-E2EP3 responses with those of patients infected with non-CHIKV alphaviruses, or flaviviruses. E2EP3 cross-reactive samples from patients infected with non-CHIKV viruses were further analyzed with an in vitro CHIKV neutralization assay. CHIKV-specific anti-E2EP3 antibody responses were detected in 72% to 100% of patients. Serum samples from patients infected with other non-CHIKV alphaviruses were cross-reactive to E2EP3. Interestingly, some of these antibodies demonstrated clearly in vitro CHIKV neutralizing activity. Contrastingly, serum samples from flaviviruses-infected patients showed a low level of cross-reactivity against E2EP3. Using CHIKV E2EP3 as a serology marker not only allows early detection of CHIKV specific antibodies, but would also allow the differentiation between CHIKV infections and flavivirus infections with 93% accuracy, thereby allowing precise acute febrile diagnosis and improving clinical management in regions newly suffering from CHIKV outbreaks including the Americas.
Author Summary
Chikungunya virus (CHIKV) causes Chikungunya fever in humans. The symptoms, particularly joint pain, can be severe and long lasting, and outbreaks can have serious socioeconomic impact. CHIKV is a mosquito-borne alphavirus that co-exists geographically with other mosquito-borne flaviviruses such as dengue virus (DENV). This causes difficulties in diagnosis because the symptoms are similar between CHIKV and DENV infections. It is important to differentiate between CHIKV and DENV infections, with good diagnostic methods. In this paper, we found that 72%–100% of CHIKV-infected patients had antibodies that recognized E2EP3, a part of a CHIKV protein. In contrast, a low percentage of flavivirus-infected patients had antibodies that recognized E2EP3. This suggests that testing patients for the presence of E2EP3-recognizing antibodies will aid in diagnostic differentiation between CHIKV and DENV infections. Interestingly, patients infected with non-chikungunya alphaviruses had moderate levels of antibodies that recognized E2EP3. While it was generally known that the alphaviruses have fairly conserved amino acid sequences, it was unknown until now, to what extent the antibodies against non-chikungunya viruses would also recognize E2EP3 from CHIKV. This paper provides insights about the E2EP3-recognizing antibodies from patients with different mosquito-borne viral infections and these insights will inform approaches to diagnostics and vaccination.
PMCID: PMC4287563  PMID: 25568956
18.  Loss of TLR3 aggravates CHIKV replication and pathology due to an altered virus-specific neutralizing antibody response 
EMBO Molecular Medicine  2014;7(1):24-41.
RNA-sensing toll-like receptors (TLRs) mediate innate immunity and regulate anti-viral response. We show here that TLR3 regulates host immunity and the loss of TLR3 aggravates pathology in Chikungunya virus (CHIKV) infection. Susceptibility to CHIKV infection is markedly increased in human and mouse fibroblasts with defective TLR3 signaling. Up to 100-fold increase in CHIKV load was observed in Tlr3−/− mice, alongside increased virus dissemination and pro-inflammatory myeloid cells infiltration. Infection in bone marrow chimeric mice showed that TLR3-expressing hematopoietic cells are required for effective CHIKV clearance. CHIKV-specific antibodies from Tlr3−/− mice exhibited significantly lower in vitro neutralization capacity, due to altered virus-neutralizing epitope specificity. Finally, SNP genotyping analysis of CHIKF patients on TLR3 identified SNP rs6552950 to be associated with disease severity and CHIKV-specific neutralizing antibody response. These results demonstrate a key role for TLR3-mediated antibody response to CHIKV infection, virus replication and pathology, providing a basis for future development of immunotherapeutics in vaccine development.
PMCID: PMC4309666  PMID: 25452586
Chikungunya virus; innate immunity; joint inflammation; neutralizing antibodies; TLR3
19.  MIF receptor CD74 mediates alphavirus-induced arthritis and myositis 
Arthritis and rheumatism  2013;65(10):2724-2736.
Arthrogenic alphaviruses such as Ross River virus (RRV) and chikungunya virus (CHIKV) circulate worldwide. This virus class causes debilitating illnesses characterised by arthritis, arthralgia, and myalgia. We previously identified macrophage migration inhibitory factor (MIF) as a critical inflammatory factor in the pathogenesis of alphaviral disease. Here, we characterise the role of CD74, a cell surface receptor of MIF, in both RRV- and CHIKV-induced alphavirus arthritides.
Mouse models of RRV and CHIKV infection were used to investigate the immunopathogenesis of arthritic alphavirus infection. The role of CD74 was assessed using histological analysis, real time PCR, flow cytometry and plaque assay.
In comparison to wild-type mice, CD74−/− mice developed only mild clinical features and had low levels of tissue damage. Leukocyte infiltration, characterised predominantly by inflammatory monocytes and natural killer cells, was substantially reduced in infected tissues of CD74−/− mice, but production of pro-inflammatory cytokines and chemokines were not decreased. CD74 deficiency was associated with increased monocyte apoptosis, but had no effect on monocyte migratory capacity. Consistent with these findings, alphaviral infection resulted in a dose-dependent up-regulation of CD74 expression in human peripheral blood mononuclear cells and serum MIF levels were significantly elevated in humans with RRV or CHIKV infections.
We propose that CD74 regulates immune responses to alphaviral infection through effects on cellular recruitment and survival. These findings suggest that both MIF and CD74 play a critical role in mediating alphaviral disease and blocking these factors with novel therapeutic agents can substantially ameliorate pathology.
PMCID: PMC3796577  PMID: 23896945
20.  Self-Reported Pain Intensity with the Numeric Reporting Scale in Adult Dengue 
PLoS ONE  2014;9(5):e96514.
Pain is a prominent feature of acute dengue as well as a clinical criterion in World Health Organization guidelines in diagnosing dengue. We conducted a prospective cohort study to compare levels of pain during acute dengue between different ethnicities and dengue severity.
Demographic, clinical and laboratory data were collected. Data on self-reported pain was collected using the 11-point Numerical Rating Scale. Generalized structural equation models were built to predict progression to severe disease.
A total of 499 laboratory confirmed dengue patients were recruited in the Prospective Adult Dengue Study at Tan Tock Seng Hospital, Singapore. We found no statistically significant differences between pain score with age, gender, ethnicity or the presence of co-morbidity. Pain score was not predictive of dengue severity but highly correlated to patients’ day of illness. Prevalence of abdominal pain in our cohort was 19%. There was no difference in abdominal pain score between grades of dengue severity.
Dengue is a painful disease. Patients suffer more pain at the earlier phase of illness. However, pain score cannot be used to predict a patient’s progression to severe disease.
PMCID: PMC4006847  PMID: 24788828
21.  Challenges in Dengue Fever in the Elderly: Atypical Presentation and Risk of Severe Dengue and Hospita-Acquired Infection 
To better understand dengue fever in the elderly, we compared clinical features, World Health Organization (WHO) dengue classification and outcomes between adult (<60) and elderly (≥60) dengue patients. We explored the impact of co-morbidity and hospital-acquired infection (HAI) on clinical outcomes in the elderly. All patients managed at the Communicable Disease Centre, Singapore, between 2005 and 2008 with positive dengue polymerase chain reaction (PCR) or who fulfilled WHO 1997 or 2009 probable dengue criteria with positive dengue IgM were included.
Of the 6989 cases, 295 (4.4%) were elderly. PCR was positive in 29%. The elderly suffered more severe disease with more dengue haemorrhagic fever (DHF) (29.2% vs. 21.4%) and severe dengue (SD) (20.3% vs. 14.6%) (p<0.05). Classic dengue symptoms were more common in the adult group. The elderly were less likely to fulfill WHO 1997 (93.6% vs. 96.4%) (p = 0.014), but not WHO 2009 probable dengue (75.3% vs. 71.5%). Time to dengue diagnosis was similar. There was no significant difference in the frequency of warning signs between the two groups, but the elderly were more likely to have hepatomegaly (p = 0.006) and malaise/lethargy (p = 0.033) while the adults had significantly more mucosal bleeding (p<0.001). Intensive care admission occurred in 15 and death in three, with no age difference. Notably, the elderly stayed in hospital longer (median 5 vs. 4 days), and suffered more pneumonia (3.8% vs. 0.7%) and urinary infection (1.9% vs. 0.3%) (p = 0.003). Predictors of excess length of stay were age (adjusted odds ratio [aOR] 2.01, 95% confidence interval [CI] 1.37–2.88), critical illness (aOR 5.13, 95%CI 2.59–9.75), HAI (aOR 12.06, 95%CI 7.39–19.9), Charlson score (aOR 6.9, 95%CI 2.02–22.56) and severe dengue (DHF/dengue shock syndrome/SD) (aOR 2.24, 95%CI 1.83–2.74).
Elderly dengue patients present atypically and are at higher risk of DHF, SD and HAI. Aside from dengue severity, age, co-morbidity and HAI were associated with longer hospital stay.
Author Summary
Dengue is a neglected tropical disease that is increasingly affecting elderly patients; however, there is a paucity of data on clinical presentation and outcomes in this group. The limited data suggests that elderly dengue patients have the highest case-fatality rate but the pathogenesis of mortality in elderly dengue patients remains unclear. To better understand dengue fever in the elderly we compared clinical features, WHO dengue classification and outcomes between adult (<60) and elderly (≥60) dengue patients and explored the impact of co-morbidity and HAI on clinical outcomes in the elderly. We found that diagnosis in the elderly may be challenging due to atypical presentation. Elderly patients have worse outcomes compared with their younger counterparts with increased rates of DHF and SD. Elderly patients have higher rates of HAI placing them at risk of infection-related mortality. Aside from dengue severity, age, co-morbidity and HAI were associated with longer hospital stay. This will place further burden on already stretched hospital systems.
PMCID: PMC3974675  PMID: 24699282
22.  Diagnosing Dengue at the Point-of-Care: Utility of a Rapid Combined Diagnostic Kit in Singapore 
PLoS ONE  2014;9(3):e90037.
WHO recommendations for dengue diagnosis require laboratory facilities. Antibody-based rapid diagnostic tests (RDTs) have performed poorly, and clinical diagnosis remains the mainstay in dengue-endemic countries. We evaluated a combination antigen-antibody RDT for point-of-care testing in a high-prevalence setting. In this prospective cohort study, adults were enrolled from a tertiary infectious disease centre for evaluation of undifferentiated febrile illness from October 2011 to May 2012. SD Bioline Dengue Duo was evaluated at point-of-care against a WHO-based reference standard of viral isolation, RT-PCR, NS1-, IgM-, and IgG-ELISA. 246 adults were enrolled (median age 34 years, range 18–69), of which 197 could be confirmed definitively as either dengue or non-dengue. DENV-2 was the predominant serotype (79.5%) and the ratio of primary to secondary cases was 1∶1.1. There were no test failures and minimal interobserver variation with a Fleiss’ kappa of 0.983 (95% CI 0.827–1.00). Overall sensitivity and specificity were 93.9% (95% CI 88.8–96.8%) and 92.0% (95% CI 81.2–96.9%) respectively. Using WHO clinical criteria alone for diagnosis had similar sensitivities (95.9%, 95% CI 91.4–98.1%) and lower specificities (20.0%, 95% CI 11.2–33.0%). No significant difference in performance was found when testing early versus late presenters, primary versus secondary cases, or DENV-1 versus DENV-2 infections. The use of a combination RDT fulfills WHO ASSURED criteria for point-of-care testing and can enhance dengue diagnosis in an endemic setting. This has the potential to markedly improve clinical management of dengue in the field.
PMCID: PMC3960091  PMID: 24646519
23.  Clinical Evaluation of a Low Cost, In-House Developed Real-Time RT-PCR Human Immunodeficiency Virus Type 1 (HIV-1) Quantitation Assay for HIV-1 Infected Patients 
PLoS ONE  2014;9(3):e89826.
HIV-1 viral quantitation is essential for treatment monitoring. An in-house assay would decrease financial barriers to access.
Materials and Methods
A real-time competitive RT-PCR in house assay (Sing-IH) was developed in Singapore. Using HXB2 as reference, the assay's primers and probes were designed to generate a 183-bp product that overlaps a portion of the LTR region and gag region. A competitive internal control (IC) was included in each assay to monitor false negative results due to inhibition or human error. Clinical evaluation was performed on 249 HIV-1 positive patient samples in comparison with the commercially available Generic HIV Viral Load assay. Correlation and agreement of results were assessed for plasma HIV-1 quantification with both assays.
The assay has a lower limit of detection equivalent to 126 copies/mL of HIV-1 RNA and a linear range of detection from 100–1000000 copies/mL. Comparative analysis with reference to the Generic assay demonstrated good agreement between both assays with a mean difference of 0.22 log10 copies/mL and 98.8% of values within 1 log10 copies/mL range. Furthermore, the Sing-IH assay can quantify HIV-1 group M subtypes A–H and group N isolates adequately, making it highly suitable for our region, where subtype B and CRF01_AE predominate.
With a significantly lower running cost compared to commercially available assays, the broadly sensitive Sing-IH assay could help to overcome the cost barriers and serve as a useful addition to the currently limited HIV viral load assay options for resource-limited settings.
PMCID: PMC3945479  PMID: 24603460
24.  Dengue Serotype Cross-Reactive, Anti-E Protein Antibodies Confound Specific Immune Memory for 1 Year after Infection 
Dengue virus has four serotypes and is endemic globally in tropical countries. Neither a specific treatment nor an approved vaccine is available, and correlates of protection are not established. The standard neutralization assay cannot differentiate between serotype-specific and serotype cross-reactive antibodies in patients early after infection, leading to an overestimation of the long-term serotype-specific protection of an antibody response. It is known that the cross-reactive response in patients is temporary but few studies have assessed kinetics and potential changes in serum antibody specificity over time. To better define the specificity of polyclonal antibodies during disease and after recovery, longitudinal samples from patients with primary or secondary DENV-2 infection were collected over a period of 1 year. We found that serotype cross-reactive antibodies peaked 3 weeks after infection and subsided within 1 year. Since secondary patients rapidly produced antibodies specific for the virus envelope (E) protein, an E-specific ELISA was superior compared to a virus particle-specific ELISA to identify patients with secondary infections. Dengue infection triggered a massive activation and mobilization of both naïve and memory B cells possibly from lymphoid organs into the blood, providing an explanation for the surge of circulating plasmablasts and the increase in cross-reactive E protein-specific antibodies.
PMCID: PMC4132268  PMID: 25177321
Dengue; viral infection; antibodies; B cells; plasmablasts; longitudinal studies; cross-reactive; vaccines
25.  Higher Risk of Infection with Dengue at the Weekend among Male Singaporeans 
A growing body of evidence suggests that dengue infection in Singapore predominantly occurs away from the home, but when and where dengue transmission occurs is unclear, confounding control efforts. The authors estimate days of the week in which dengue inpatients in Singapore were infected during the period 2006–2008, based on the day they became febrile and historical data on the incubation period, using Bayesian statistical methods. Among male inpatients, the relative risk of infection is an estimated 57% higher at the weekend, suggesting infections associated with the home or leisure activities. There was no evidence of elevated risk of infection at the weekend for female inpatients. The study motivates further research identifying locales frequented in the week leading up to onset to improve the effective targeting of vector control efforts.
PMCID: PMC3516085  PMID: 23128291

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